Oxidized fungal antigens and methods of making and using thereof

ABSTRACT

The present invention relates to oxidized fungal antigens and methods of making and using thereof. More particularly, the present invention provides a method for producing an oxidized fungal antigen in culture filtrate. The present invention also provides for the produced oxidized fungal antigens. Devices comprising such oxidized fungal antigens, methods for testing for fungal antibodies using the oxidized fungal antigens and methods for producing anti-fungal antibodies using oxidized fungal antigens are further provided. Antigen detection devices comprising anti-fungal antibodies raised against oxidized fungal antigens produced by the present methods are further provided.

TECHNICAL FIELD

[0001] The present invention relates to oxidized fungal antigens andmethods of making and using thereof. More particularly, the presentinvention provides a method for producing an oxidized fungal antigen inculture filtrate. The present invention also provides for the producedoxidized fungal antigens. Devices comprising such oxidized fungalantigens, methods for testing for fungal antibodies using the oxidizedfungal antigens and methods for producing anti-fungal antibodies usingoxidized fungal antigens are further provided. Antigen detection devicescomprising anti-fungal antibodies raised against oxidized fungalantigens produced by the present methods are further provided.

BACKGROUND ART

[0002] Fungal infections are common in the United States, and are oftenassociated with significant morbidity. For example, Candida species arethe fourth most common organisms isolated from US hospital patients(Wenzel and Pfaller, Infect. Control Hosp. Epidemiol., 12:523-524(1991)). Histoplasma capsulatum and Coccidioides immitis causes,respectively, 250,000 and 100,000 new infections annually, and 10-40% ofinfected persons become symptomatic (Bullock, Histoplasma capsulatum,In: Principles and Practice of Infectious Diseases, G L Mandell et al.(eds.), Churchill Livingstone, N.Y., 1995, pp. 2340-2353 and 2365-2375;and Stevens, N. Engl. J. Med., 332:1077-1082 (1995)). Individuals withsymptomatic C. immitis infection miss an average of 35 days of school orwork, and incur a total of $24 million in medical expenses annually.

[0003] Many fungi cause respiratory infections that areindistinguishable on the basis of symptoms. However, it is important toaccurately determine which fungus is responsible for infection due todifferences in treatment regimens and incidences of complications(Bullock, Histoplasma capsulatum, In: Principles and Practice ofInfectious Diseases, G L Mandell et al. (eds.), Churchill Livingstone,N.Y., 1995, pp. 2340-2353 and 2365-2375; and Galgiani, Ann. Intern.Med., 130:293-300 (1999)). Because culture and direct antigen detectionare problematic, antibody detection plays an important role in thediagnosis and identification of fungal infections. The methods mostcommonly used to detect fungal antibodies include complement fixation(CF) and immunodiffusion (ID) (Zancope-Oliveira et al., Clin. Diagn.Lab. Immunol., 1:90-93 (1994)). Both methods, however, have inherentdrawbacks. ID detects antibodies to species-specific protein moieties,and is thus sensitive and highly specific; however, the assay takes 48hours to perform, and appropriate interpretation requires highly skilledpersonnel. Although CF assays are highly sensitive, their performance iscomplex and labor-intensive, and they exhibit low specificity due tocross-reactive antibodies recognizing carbohydrate moieties common toseveral fungi (Zancope-Oliveira et al., Clin. Diagn. Lab. Immunol.,1:90-93 (1994); and Yang et al., Clin. Diagn. Lab. Immunol., 4:19-22(1997)). Enzyme-linked immunosorbent assays (ELISA) for the detection offungal antibodies have also been described, but these assays exhibit lowspecificity due to the same cross-reactive antibodies at issue in CFassays (Zartarian et al., Am. J. Clin. Pathol., 207:148-153 (1997); andKaufman et al., J. Clin. Microbiol., 33:618-619 (1995)).

[0004] During the last decade, investigators using a western blot assaysystem found that periodate oxidation of fungal antigen preparationsinactivates the cross-reactive carbohydrate moieties but does notdisturb the structural integrity of the protein moieties(Zancope-Oliveira et al., Clin. Diagn. Lab. Immunol, 1:90-93 (1994);Zancope-Oliveira et al., Clin. Diagn. Lab. Immunol., 1:563-568 (1994);and Pizzini et al., Clin. Diagn. Lab. Immunol., 6:20-23 (1999)). Thus,utilization of oxidized fungal antigens markedly increased thespecificity of the western blot assay for fungal antibodies, withoutaffecting sensitivity. However, the western blot assay described thereinis not practical for most medical laboratories; many complex steps arerequired, including antigen preparation, gel electrophoresis,electrotransfer to nitrocellulose membranes, assay performance, and blotinterpretation. The antigen was a culture filtrate, 0.45 μm filtered,20× concentrated by ultrafiltration, dialyzed against PBS, purified byion exchange chromatography, 10× concentrated by ultrafiltration, andfinally dialyzed against PBS. Oxidizing the antigen also includedseveral labor intensive and potentially contaminating steps: 4 mg/mLantigen was exposed to periodate for 18 hours at 4° C., treated withglycerol for 15 minutes, treated with borohydrate for 2 hours, andfinally dialyzed against water. Manufacturing the devices also includedmethods requiring sophisticated equipment and skilled technicians:denaturing the oxidized antigen with heat and mercaptoethanol,electrophoresing with a 4% stacking gel and 7.5% resolving gel,transferring the proteins to nitrocellulose paper, blocking withbuffered milk and tween, drying the paper, and cutting the paper intostrips, etc.

[0005] Although attempts to utilize periodate-treated fungal antigensfor specific detection of fungal antibodies in an ELISA system have beenreported, these attempts were only marginally successful (Fisher et al.,Mycroses, 40:83-90 (1997)). Binding of H. capsulatum antibodies tooxidized Blastomyces dermatitidis antigens was reduced only by about 20%when compared to binding to non-oxidized B. dermatitidis antigens. Thislimited reduction in cross-reactivity may have been due to the use ofhighly purified (and thus extensively manipulated) fungal antigens,periodate oxidation after the antigens had adsorbed to the plasticsurface, or the use of an insufficient concentration of periodate.

[0006] Therefore, there exists a need in the art for oxidized fungalantigens with more reduced cross-reactivity. The present inventionaddresses this and other related needs.

DISCLOSURE OF THE INVENTION

[0007] In one aspect, the present invention is directed to a method forproducing a fungal antigen suitable for testing for an antibody to afungus, the method comprising: a) providing a fungal antigen as aculture filtrate, the fungal antigen having not been purified by ionexchange chromatography or isoelectric focusing from the culturefiltrate; and b) contacting the fungal antigen with an oxidizing agentto produce an oxidized fungal antigen suitable for testing for anantibody to the fungus. Devices comprising an oxidized fungal antigen,or a plurality of oxidized fungal antigens, produced by the abovemethod, for testing for an anti-fungal antibody, or a plurality ofanti-fungal antibodies, are also provided.

[0008] In another aspect, the present invention is directed to a methodof testing for an antibody to a fungus in a sample, the methodcomprising: a) producing a fungal antigen suitable for testing for anantibody to a fungus, comprising providing a fungal antigen as a culturefiltrate, the fungal antigen having not been purified by ion exchangechromatography or isoelectric focusing from the culture filtrate, andcontacting the fungal antigen with an oxidizing agent to produce anoxidized fungal antigen suitable for testing for an antibody to thefungus; b) contacting a sample suspected of containing an antibody to afungus with the oxidized fungal antigen produced in step a) undersuitable conditions to allow binding of the antibody, if present in thesample, to the oxidized fungal antigen; and c) assessing binding betweenthe antibody and the oxidized fungal antigen to determine the presenceand/or amount of the antibody in the sample.

[0009] In still another aspect, the present invention is directed to amethod for producing an antibody to a fungal antigen, the methodcomprising: a) producing a fungal antigen comprising providing a fungalantigen as a culture filtrate, the fungal antigen having not beenpurified by ion exchange chromatography or isoelectric focusing from theculture filtrate, and contacting the fungal antigen with an oxidizingagent to produce an oxidized fungal antigen; b) delivering, to avertebrate, the oxidized fungal antigen, in an amount sufficient toinduce detectable production of an antibody to the antigen; and c)recovering the antibody from the vertebrate.

[0010] In yet another aspect, the present invention is directed to amethod for producing a monoclonal antibody to a fungal antigen, themethod comprising: a) producing a fungal antigen comprising providing afungal antigen as a culture filtrate, the fungal antigen having not beenpurified by ion exchange chromatography or isoelectric focusing from theculture filtrate, and contacting the fungal antigen with an oxidizingagent to produce an oxidized fungal antigen; b) delivering, to avertebrate, the oxidized fungal antigen, in an amount sufficient toinduce detectable production of an antibody to the antigen; c) removingat least a portion of antibody-producing cells from the vertebrate; d)immortalizing the removed antibody-producing cells; e) propagating theimmortalized antibody-producing cells; and f) harvesting monoclonalantibody produced by the immortalized antibody-producing cells. Methodsof testing for a fungal antigen in a sample using the producedantibodies are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1 illustrates a Histoplasma capsulatum IgG ELISA test usingan oxidized and a non-oxidized antigen.

[0012]FIG. 2 illustrates a Coccidioides immitis IgG ELISA test using anoxidized and a non-oxidized antigen.

[0013]FIG. 3 illustrates a Aspergillus fumigatus IgG ELISA test using anoxidized and a non-oxidized antigen.

[0014]FIG. 4 illustrates a Blastomyces dermatitidis IgG ELISA test usingan oxidized and a non-oxidized antigen.

MODES OF CARRYING OUT THE INVENTION

[0015] For clarity of disclosure, and not by way of limitation, thedetailed description of the invention is divided into the subsectionsthat follow.

[0016] A. Definitions

[0017] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as is commonly understood by one ofordinary skill in the art to which this invention belongs. All patents,applications, published applications and other publications referred toherein are incorporated by reference in their entirety. If a definitionset forth in this section is contrary to or otherwise inconsistent witha definition set forth in the patents, applications, publishedapplications and other publications that are herein incorporated byreference, the definition set forth in this section prevails over thedefinition that is incorporated herein by reference.

[0018] As used herein, “a” or “an” means “at least one” or “one ormore.”

[0019] As used herein, “fungus” refers to a division of eucaryoticorganisms that grow in irregular masses, without roots, stems, orleaves, and are devoid of chlorophyll or other pigments capable ofphotosynthesis. Each organism (thallus) is unicellular to filamentous,and possesses branched somatic structures (hyphae) surrounded by cellwalls containing glucan or chitin or both, and containing true nuclei.

[0020] As used herein, “culture” refers to a growth of living cells ormicroorganisms, e.g., fungi, in a controlled artificial environment.

[0021] As used herein, “filtration” refers to a process of separatingparticulate matter from a fluid, such as air or liquid, by passing thefluid carrier through a medium that will not pass the particulates.

[0022] As used herein, “filtrate” refers to a fluid, e.g., a liquid,that passes through the medium in filtration.

[0023] As used herein, “a fungal antigen as a culture filtrate” refersto a culture filtrate of a fungus containing the fungal antigenicsubstances. The culture filtrate can contain secreted extracellularfungal antigenic substances derived from supernatant of the fungalculture. Alternatively, culture filtrate can contain intracellularfungal antigenic substances derived from solution or suspension ofdisrupted fungal cells.

[0024] As used herein, “fungal antigen having (or has) not beenpurified” means that a particular fungal antigen (or a group of fungalantigens) has not been isolated or purified from other antigenicsubstances in the culture filtrate by any isolation or purificationmethods. However, the fungal antigen existing in the culture filtratecan be concentrated or diluted along with the concentration or dilutionof the culture filtrate.

[0025] As used herein, “fungal antigen having not been purified by ionexchange chromatography or isoelectric focusing from the culturefiltrate” means that a particular fungal antigen (or a group of fungalantigens) has not been isolated or purified from other antigenicsubstances in the culture filtrate by ion exchange chromatography orisoelectric focusing. However, the fungal antigen existing in theculture filtrate can be concentrated or diluted along with theconcentration or dilution of the culture filtrate, or can be isolated orpurified from other antigenic substances in the culture filtrate by anyisolation or purification methods other than ion exchange chromatographyand isoelectric focusing.

[0026] As used herein, an “oxidizing agent (or oxidant)” refers to anagent that accepts electrons in an oxidation-reduction reaction.

[0027] As used herein the term “assessing (or assessed)” is intended toinclude quantitative and qualitative determination of the identity of amoiety, e.g. an antigen, an antibody or an antigen-antibody complex,present in the sample, and also of obtaining an index, ratio,percentage, visual or other value indicative of the identity of a moietyin the sample. Assessment may be direct or indirect.

[0028] As used herein, “sample” refers to anything which may contain afungal antigen or antibody to be tested by the present devices and/ormethods. The sample may be a biological sample, such as a biologicalfluid or a biological tissue. Examples of biological fluids includeurine, blood, plasma, serum, saliva, semen, stool, sputum, cerebralspinal fluid, tears, mucus, amniotic fluid or the like. Biologicaltissues are aggregates of cells, usually of a particular kind togetherwith their intercellular substance that form one of the structuralmaterials of a human, animal, plant, bacterial, fungal or viralstructure, including connective, epithelium, muscle and nerve tissues.Examples of biological tissues also include organs, tumors, lymph nodes,arteries and individual cell(s). The sample may also be a fungalculture, e.g., a fungal culture derived from a biological sample.

[0029] As used herein, a “liquid (fluid) sample” refers to a sample thatnaturally exists as a liquid or fluid, e.g., a biological fluid. A“liquid sample” also refers to a sample that naturally exists in anon-liquid status, e.g., solid or gas, but is prepared as a liquid,fluid, solution or suspension containing the solid or gas samplematerial. For example, a liquid sample can encompass a liquid, fluid,solution or suspension containing a biological tissue.

[0030] B. Methods for Producing Oxidized Fungal Antigen

[0031] In one aspect, the present invention is directed to a method forproducing a fungal antigen suitable for testing for an antibody to afungus, the method comprising: a) providing a fungal antigen as aculture filtrate, the fungal antigen having not been purified by ionexchange chromatography or isoelectric focusing from the culturefiltrate; and b) contacting the fungal antigen with an oxidizing agentto produce an oxidized fungal antigen suitable for testing for anantibody to the fungus.

[0032] The present methods can be used to produce an oxidized fungalantigen from any fungal genus or species. The oxidized antigens, whilebeing reactive to antibodies to the fungus from which the fungal antigenis derived, may be reactive to antibodies to the fungus of other relatedgenus or species. Preferably, the methods will be controlled to produceoxidized fungal antigens that are able to distinguish among differentfungal genus. More preferably, the methods will be controlled to produceoxidized fungal antigens that are able to distinguish among differentfungal species within the same genus.

[0033] Fungi can be generally described as follows (Brock and Madigan,Biology of Microorganisms (6th Ed.), Prentice Hall, Englewood Cliffs,N.J. 07632 (1991), pp. 817-818). In contrast to the algae, the fungilack chlorophyll. Fungi can be differentiated from bacteria by the factthat fungal cells are usually much larger and contain a nucleus,vacuoles, and mitochondria, typical of eucaryotic cells. Although thefungi are a large and diverse group of eucaryotic microorganisms, threegroups of fungi have major practical importance: the molds, yeasts, andmushrooms.

[0034] The habitats of fungi are quite diverse. Some are aquatic, livingprimarily in fresh water, and a few marine fungi are also known. Mostfungi, however, have terrestrial habitats, in soil or on dead plantmatter, and these types often play crucial roles in the mineralizationof organic carbon in nature. A large number of fungi are parasites ofterrestrial plants. Indeed, fungi cause the majority of economicallysignificant diseases of crop plants (see Table 1). A few fungi areparasitic on animals, including humans, although in general fungi areless significant as animal pathogens than are bacteria and viruses.TABLE 1 Classification and major properties of fungi Common Typical Typeof Common Group name Hyphae representatives sexual spore Habitatsdiseases Ascomycetes Sac fungi Septate Neurospora, Ascospore Soil, Dutchelm, Saccharomyces, decaying chestnut blight, Morchella plant ergot,rots (morels) material Basidiomycetes Club fungi, Septate AmanitaBasidiospore Soil, Black stem, mushrooms (poisonous decaying wheat rust,mushroom), plant corn smut Agaricus material (edible mushroom)Zygomycetes Bread Coenocytic Mucor, Zygospore Soil, Food spoilage; moldsRhizopus decaying rarely involved (common plant in parasitic bread mold)material disease Oomycetes Water Coenocytic Allomyces Oospore AquaticPotato blight, molds certain fish diseases Deuteromycetes Fungi SeptatePenicillium, None Soil, Plant wilt, imperfecti Aspergillus decayingfungal plant infections of material, animals such as surfaces ofringworm, animal athlete's foot, bodies and other dermatomycoses

[0035] All fungi are organotrophs. Lacking chlorophyll, they of coursecannot photosynthesize, and the group also lacks lithotrophic forms.When compared to the bacteria, the fungi in general have fairly simplenutritional requirements, and their metabolic and biosynthetic processesare not particularly diverse or unusual. It is in their morphologicalproperties and in their sexual life cycles that the fungi exhibitconsiderable diversity; hence it is on the basis of thesecharacteristics that the fungi are classified.

[0036] In a specific embodiment, the present method is used to producean oxidized fungal antigen from a mitosporic Trichocomaceae, anOnygenaceae or a mitosporic Onygenale. In another specific embodiment,the present method is used to produce an oxidized fungal antigen fromAspergillus, e.g., Aspergillus fumigatus, Blastomyces (Ajellomyces),e.g., Blastomyces dermatitidis, Coccidioides, e.g., Coccidioidesimmitis, Blastocystis, e.g., Blastocystis hominis, Histoplasma, e.g.,Histoplasma capsulatum and Histoplasma duboifii, Candida, e.g., Candidaalbicans and Cyrptococcus, e.g., Cyrptococcus neoformans.

[0037] In still another specific embodiment, the present method is usedto produce an oxidized fungal antigen from a fungal antigen not derivedfrom Aspergillus, e.g., Aspergillus fumigatus, Blastomyces(Ajellomyces), e.g., Blastomyces dermatitidis, Coccidioides, e.g.,Coccidioides immitis, Blastocystis, e.g., Blastocystis hominis,Histoplasma, e.g., Histoplasma capsulatum and Histoplasma duboifii,Candida, e.g., Candida albicans and Cyrptococcus, e.g., Cyrptococcusneoformans.

[0038] The fungal antigen to be oxidized should not have been purifiedby ion exchange chromatography or isoelectric focusing from the culturefiltrate. However, the fungal antigen to be oxidized can be concentratedor diluted along with the concentration or dilution of the culturefiltrate, or can be isolated or purified from other antigenic substancesin the culture filtrate by any isolation or purification methods otherthan ion exchange chromatography and isoelectric focusing. For example,the fungal antigen can be isolated or purified by any chromatographicmethods other than ion exchange chromatography or any electrophoresisother than isoelectric focusing, as well as other types of isolation orpurification methods such as centrifugation or organic-aqueous phaseseparations. In a specific embodiment, the fungal antigen to be oxidizedhas not been purified from the culture filtrate prior to the oxidationstep.

[0039] The present methods can comprise additional steps. For example,the present methods can further comprise a step of concentrating theculture filtrate. The concentrating step can be conducted prior to,concurrently with and/or subsequent to the oxidation step. Preferably,the concentrating step is conducted prior to the oxidation step. Inanother example, the present methods can further comprise a step ofenzymatically deglycosylating the fungal antigen. Any suitabledeglycosylating enzymes can be used, e.g., Endo H, PNGase F,O-glycancase, etc. The enzymatic deglycosylation step can be conductedprior to, concurrently with and/or subsequent to the oxidation step.Preferably, enzymatic deglycosylation step is conducted prior to theoxidation step.

[0040] Any suitable oxidizing agent can be used in the present methods.Exemplary oxidizing agents include hydrogen peroxide (H₂H₂), ozone (O₃),polyatomic oxygen O₇, polyatomic oxygen O₈, NaIO₄, potassiumperoxymonosulfate (oxone) (Wozniak et al., Bioorg. Med. Chem. Lett.,8(19):2641-6 (1998)), D,L-S-methyllipoic acid methyl ester (Pan andJordan, Biochemistry, 37(5):1357-64 (1998)), tertiary butylhydroperoxide (Tarin et al., Mol. Hum. Reprod., 2(12):895-901 (1996)),menadione (Santini et al., Free Radic. Biol. Med., 20(7):915-24 (1996)),diamide (Bosin and Kasper, J. Biochem. Toxicol., 7(3):139-45 (1992)),iodogen (Saha et al., Int. J. Rad. Appl. Instrum., 16(4):431-3 (1989)),N-bromosuccinimide (Sinn et al., Anal. Biochem., 170(1):186-92 (1988)),omeprazole (Im et al., J. Biol. Chem., 260(8):4591-7 (1985)), andN-ethylmaleimide (Marzulli et al., Boll. Soc. Ital. Biol. Sper.,61(1):121-7 (1985)).

[0041] In a specific embodiment, the oxidizing agent used in the presentmethods inactivates the cross-reactive carbohydrate moiety of the fungalantigen but does not disturb the structural integrity or antigenicity ofthe non-carbohydrate moieties. For example, when the fungal antigen tobe oxidized is a glycosylated protein or peptide, the oxidizing agentinactivates the cross-reactive carbohydrate moiety of the fungal antigenbut does not disturb the structural integrity or antigenicity of theproteineous or peptidyl moiety.

[0042] In a preferred embodiment, the oxidizing agent used in thepresent methods is periodate (or periodic acid), which has a molecularformula H₅IO₆. Suitable salts, esters or other derivatives of periodatethat retain periodate's oxidizing activity, e.g., sodium meta-periodate(Sigma), can also be used. Periodate or its salts, esters or otherderivatives thereof can be produced by methods known in the art. Forexample, periodate can be prepared by electrolytic oxidation of iodicacid or from barium periodate and nitric acid (Willard, Inorg. Syn. 1,172 (1939); Chemistry of periodic acid and periodates; H. Siebert,Fortschr. Chem. Forsch. 8, 470 (1967); Periodic acid and periodates inorganic and bioorganic chemistry; A. J. Fatiadi, Synthesis 1974, 229;and G. Dryhurst, Periodate Oxidation of Diol and Other Functional Groups(Pergamon Press, New York, 1970)). Alternatively, Periodate or itssalts, esters or other derivatives thereof can be obtained commercially,e.g., from Sigma.

[0043] The oxidation step can be conducted under any suitable conditionsdepending on a number of factors such as the fungal antigen to beoxidized, the oxidizing agent to be used, the properties of the culturefiltrate, the degree of concentration and/or dilution of the culturefiltrate and the presence or absence of any enzymatic deglycosylation.Generally, the concentration of the oxidizing agent can be in a widerange, e.g., from 0.01 M to about 0.1 M if periodate is used. Theoxidation step can be conducted at any suitable temperature, e.g., fromabout 4° C. to an ambient temperature, e.g. from about 25° C. to about30° C. The incubation time can also have a wide range, e.g. from about 1hour to about 20 hours. When periodate is used, oxidation step ispreferably conducted at about 25° C. for about 1-3 hours.

[0044] In another specific embodiment, the present method can furthercomprise a step of attaching the oxidized fungal antigen to a surface ofa device suitable for testing for an antibody to a fungus. Any suitabledevice can be used such as a microtiter plate, a glass slide, anitrocellulose membrane, a latex bead, a cell, a test tube, a plasticbead, a colloidal gold particle, a colored particle, a magnetic bead anda quantum dot. Preferably, a microtiter plate is used.

[0045] Fungal antigens, suitable for testing for an antibody to afungus, which are produced by the present methods are also provided.Devices, suitable for testing for an antibody to a fungus, which areproduced by the present methods are further provided. In a preferredembodiment, a device for simultaneously testing for a plurality offungal antibodies is provided, the device comprising a plurality offungal antigens produced by the present methods attached to a surface ofthe device suitable for testing for an antibody to a fungus, wherein theplurality of fungal antigens are attached to areas of the surface thatare physically distinct from each other. Any suitable device can beused. In one example, the device comprises an immunoblot and wherein theplurality of fungal antigens are attached to separate stripes in theimmunoblot. In another example, the device comprises an IFA well andwherein the plurality of fungal antigens are attached to different spotswithin the IFA well. In still another example, the device comprises anIFA slide and wherein the plurality of fungal antigens are attached todifferent wells of the IFA slide. Other exemplary devices include amicrotiter plate, a glass slide, a nitrocellulose membrane, a latexbead, a cell, a test tube, a plastic bead, a colloidal gold particle, acolored particle, a magnetic bead and a quantum dot.

[0046] C. Methods of Testing for an Antibody to a Fungus

[0047] In another aspect, the present invention is directed to a methodof testing for an antibody to a fungus in a sample, the methodcomprising: a) producing a fungal antigen suitable for testing for anantibody to a fungus, comprising providing a fungal antigen as a culturefiltrate, the fungal antigen having not been purified by ion exchangechromatography or isoelectric focusing from the culture filtrate, andcontacting the fungal antigen with an oxidizing agent to produce anoxidized fungal antigen suitable for testing for an antibody to thefungus; b) contacting a sample suspected of containing an antibody to afungus with the oxidized fungal antigen produced in step a) undersuitable conditions to allow binding of the antibody, if present in thesample, to the oxidized fungal antigen; and c) assessing binding betweenthe antibody and the oxidized fungal antigen to determine the presenceand/or amount of the antibody in the sample.

[0048] The present methods can be used to test for an antibody to anyfungus in a sample, including any fungus described in the above SectionB. Preferably, the antibody to be tested is to a pathogenic fungus suchas Aspergillus, e.g., Aspergillus fumigatus, Blastomyces (Ajellomyces),e.g., Blastomyces dermatitidis, Coccidioides, e.g., Coccidioidesimmitis, Blastocystis, e.g., Blastocystis hominis, Histoplasma, e.g.,Histoplasma capsulatum and Candida, e.g., Candida albicans and Candidaneoformans.

[0049] The present methods can be used to test any sample. Preferably,the sample to be tested is a clinical sample including human andveterinary clinical samples. More preferably, the sample to be tested isa human clinical sample.

[0050] The oxidized fungal antigens to be used in the present testmethods are produced as described in the above Section B. Preferably,the fungal antigen to be oxidized has not been purified from the culturefiltrate. Also, preferably, the oxidizing agent to be used is periodate.

[0051] The present test methods can be conducted in liquid or solidphase. In specific embodiment, the present test method is conducted in asolid phase the method further comprises a step of attaching theoxidized fungal antigen to a surface of a device suitable for testingfor an antibody to a fungus before contacting the antigen with thesample.

[0052] The binding between the antibody and the oxidized fungal antigencan be assessed by any suitable assay formats. For example, the bindingbetween the antibody and the oxidized fungal antigen can be assessed bya sandwich or competitive assay format. In another example, the bindingbetween the antibody and the oxidized fungal antigen can be assessed byan enzyme-linked immunosorbent assay (ELISA), immunoblotting,immunoprecipitation, radioimmunoassay (RIA), immunostaining, latexagglutination, indirect hemagglutination assay (IHA), complementfixation, indirect immunofluorescent assay (IFA), nephelometry, flowcytometry assay, chemiluminescence assay, lateral flow immunoassay,u-capture assay, inhibition assay or avidity assay (Manual of ClinicalLaboratory Immunology, N R Rose, E Conway de Macario, J D Folds, H CLane, R M Nakamura, eds. ASM Press, Washington D.C., 1997). Preferably,the binding between the antibody and the oxidized fungal antigen isassessed by an ELISA format.

[0053] The present assay method can be conducted for prognosis,diagnosis and/or monitoring treatment of pathogenic fungal infection,e.g., histoplasmosis caused by Histoplasma capsulatum infection,blastomycosis caused by Blastomyces dermatitidis infection, candidasiscaused by Candida albicans or Candida neoformans infection, etc.

[0054] D. Methods for Producing an Antibody to an Fungal Antigen

[0055] In still another aspect, the present invention is directed to amethod for producing an antibody to a fungal antigen, the methodcomprising: a) producing a fungal antigen comprising providing a fungalantigen as a culture filtrate, the fungal antigen having not beenpurified by ion exchange chromatography or isoelectric focusing from theculture filtrate, and contacting the fungal antigen with an oxidizingagent to produce an oxidized fungal antigen; b) delivering, to avertebrate or tissue culture, the oxidized fungal antigen, in an amountsufficient to induce detectable production of an antibody to theantigen; and c) recovering the antibody from the vertebrate or tissueculture.

[0056] The oxidized fungal antigens to be used in the present antibodyproducing methods are produced as described in the above Section B.Preferably, the fungal antigen to be oxidized has not been purified fromthe culture filtrate. Also, preferably, the oxidizing agent to be usedis periodate.

[0057] Any suitable non-human mammal can be used in the present methods.For example, mouse, rabbit and goat can be used.

[0058] The antibody to a fungal antigen, e.g., a polyclonal antiserum,which is produced by the present method is also provided.

[0059] In yet another aspect, the present invention is directed to amethod for producing a monoclonal antibody to an fungal antigen, themethod comprising: a) producing a fungal antigen comprising providing afungal antigen as a culture filtrate, the fungal antigen having not beenpurified by ion exchange chromatography or isoelectric focusing from theculture filtrate, and contacting the fungal antigen with an oxidizingagent to produce an oxidized fungal antigen; b) delivering, to avertebrate or tissue culture, the oxidized fungal antigen, in an amountsufficient to induce detectable production of an antibody to theantigen; c) removing at least a portion of antibody-producing cells fromthe vertebrate or tissue culture; d) immortalizing the removedantibody-producing cells; e) propagating the immortalizedantibody-producing cells; and f) harvesting monoclonal antibody producedby the immortalized antibody-producing cells. Methods of testing for afungal antigen in a sample using the produced monoclonal antibodies arealso provided.

[0060] The oxidized fungal antigens to be used in the present antibodyproducing methods are produced as described in the above Section B.Preferably, the fungal antigen to be oxidized has not been purified fromthe culture filtrate. Also, preferably, the oxidizing agent to be usedis periodate.

[0061] Any suitable non-human mammal can be used in the present methods.For example, mouse, rabbit and goat can be used.

[0062] A monoclonal antibody to a fungal antigen, which is produced bythe present method is also provided.

[0063] A hybridoma capable of producing a monoclonal antibody to afungal antigen, which is produced by steps a)-d) of the present methodis further provided.

[0064] The oxidized fungal antigens can be delivered to a vertebrate ornon-human mammal by any methods known in the art (See e.g., Coligan etal. (Ed.), Current Protocols in Immunology, 2.II Production ofAntibodies, John Wiley & Sons, Inc. (2000)).

[0065] The oxidized fungal antigens can be delivered to the interstitialspace of tissues of the animal body, including those of muscle, skin,brain, lung, liver, spleen, bone marrow, thymus, heart lymph, blood,bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine,testis, ovary, uterus, rectum, nervous system, eye, gland, andconnective tissue. Interstitial space of the tissues comprises theintercellular, fluid, mucopolysaccharide matrix among the reticularfibers or organ tissues, elastic fibers in the walls of vessels orchambers, collagen fibers of fibrous tissues, or that same matrix withinconnective tissue ensheathing muscle cells or in the lacunae of bone. Itis similarly the space occupied by the plasma of the circulation of thelymph fluid of the lymphatic channels.

[0066] The oxidized fungal antigens can be conveniently delivered byinjection into the tissues comprising these cells. They are preferablydelivered to persistent, non-dividing cells which are differentiated,although delivery can be achieved in non-differentiated or lesscompletely differentiated cells, such as, for example, stem cells ofblood or skin fibroblasts.

[0067] In a specific embodiment, the oxidized fungal antigen isdelivered directly to a tissue of the animals. Preferably, the oxidizedfungal antigen is delivered directly to muscle, skin or mucous membrane.In one example, the oxidized fungal antigen can be delivered directly toa tissue of the animal by injection, by gene gun technology or by lipidmediated delivery technology. The injection can be conducted via aneedle or other injection devices. The gene gun technology is disclosedin U.S. Pat. No. 5,302,509 and the lipid mediated delivery technology isdisclosed in U.S. Pat. No. 5,703,055, the contents of which areincorporated herein by reference.

[0068] In still another specific embodiment, the oxidized fungal antigenis delivered to a cell of the animal and the cell containing theoxidized fungal antigen is delivered to a suitable tissue of the animal.Preferably, the oxidized fungal antigen is delivered to a blood cell ofan animal. More preferably, the oxidized fungal antigen is delivered toa spleen B cell of an animal.

[0069] The anti-fungal antibodies, whether polyclonal or monoclonal,that are produced by the above methods can be used in testing for afungal antigen in a sample. In one specific embodiment, the presentinvention is directed to a method of testing for a fungal antigen in asample, the method comprising: a) providing an anti-fungal polyclonalantiserum produced by the above antibody producing method; b) contactinga sample suspected of containing a fungal antigen with the polyclonalantiserum under suitable conditions to allow binding of the fungalantigen, if present in the sample, to the polyclonal antiserum; and c)assessing binding between the fungal antigen and the polyclonalantiserum to determine the presence and/or amount of the fungal antigenin the sample.

[0070] In another specific embodiment, the present invention is directedto a method of testing for a fungal antigen in a sample, the methodcomprising: a) providing an anti-fungal antigen monoclonal antibodyproduced by the above antibody producing method; b) contacting a samplesuspected of containing a fungal antigen with the monoclonal antibodyunder suitable conditions to allow binding of the fungal antigen, ifpresent in the sample, to the monoclonal antibody; and c) assessingbinding between the fungal antigen and the monoclonal antibody todetermine the presence and/or amount of the fungal antigen in thesample.

[0071] E. Exemplary Embodiments

[0072] In exemplary embodiments of the present invention, oxidizedpreparations of crude fungal antigens are used in an ELISA format,resulting in the sensitive and specific detection of fungal antibodies.The fungal antigens are oxidized during a short (2-hour) incubation withperiodate, then further diluted with coating buffer and allowed toadsorb to polystyrene microtiter wells. ELISA performance using thesecoated microtiter wells is complete within 4 hours, thus providingsensitive and specific results more quickly and easily than ID andwestern blot assays.

[0073] Preparation of Antigen-Coated Microtiter Wells

[0074] A fungal antigen preparation, designed for use in ID assays, ispurchased from a commercial source. The protein concentration of theantigen preparation is determined by protein-dye binding (Bradford)assay. The fungal antigen is then diluted to a final proteinconcentration of about 10-50 micrograms per milliliter (ug/mL) in 0.05Msodium acetate buffer (adjusted to pH 5.5 using 10% acetic acid)containing about 0.02M-0.10M sodium meta-periodate. After a shortincubation period (1-3 hours) at room temperature, during which hydroxylgroups of the fungal antigen carbohydrate moieties are cleaved andconverted to aldehydes (i.e., become oxidized), the oxidized fungalantigen preparation is diluted approximately 20-fold with phosphatebuffered saline (PBS), pH 7.4. This diluted fungal antigen is then addedto polystyrene microtiter wells at a volume of 0.1 mL per well. Themicrotiter wells are covered with plastic adhesive tape and incubated inthe refrigerator (4-8° C.) for 14-18 hours. During this incubation,fungal antigen protein moieties attach to the polystyrene throughhydrophobic interactions. After incubation, the solution is discardedfrom the microtiter wells, now bearing attached oxidized fungal antigen.All microtiter wells then receive 0.2 mL of PBS containing a blockingagent, such as bovine serum albumin (BSA), and a stabilizing agent, suchas trehalose. The blocking agent attaches to any polystyrene bindingsites that are left exposed after the incubation with oxidized fungalantigen. This step reduces the nonspecific attachment of antibodymolecules not directed toward the specific antigens, and also reducesnonspecific adsorption of the conjugate during the color generationstep. After 2 hours at room temperature, the blocking solution isdiscarded from the microtiter wells. The microtiter plates are air-driedat room temperature, then placed inside a plastic pouch, and the pouchheat-sealed. Sealed pouches containing microtiter plates coated withfungal antigens are stored in the refrigerator until use in the ELISA.

[0075] ELISA Performance

[0076] Antigen-coated microtiter well plates are removed from therefrigerator and allowed to warm to room temperature. The wells are thenfilled with PBS containing a mild detergent such as Tween 20 (PBST), andafter 5 minutes this PBST is discarded. This wash step ensures that allantigen and blocking solution proteins have been removed; in addition,the small amount of detergent helps to reduce bubble formation insubsequent steps. Serum samples are diluted in PBST that contains thesame protein that is used as a blocking agent (such as BSA). About 0.1mL of each diluted serum is added to an assigned microtiter well. Themicrotiter plate is then covered with adhesive tape and incubated about1.5 hours at room temperature. During this incubation period, any serumantibodies directed to the antigen that is attached to the microtiterwell will bind to the antigen. At the end of this incubation, the liquidmaterial is discarded from all the wells. About 0.25 mL of PBST is thenadded to each well, and this fluid is then discarded; this wash step isrepeated three times. This washing procedure ensures that non-attachedserum components are removed from the microtiter well. About 0.1 mL of aconjugate diluted in PBST is then added to each well. This conjugate isa commercially-available, species-specific, anti-immunoglobulin with anenzyme chemically linked (conjugated) to it. The enzyme used is selectedbased on its ability to react with a substrate to produce a coloredcompound. A typical conjugate would be goat anti-human IgG tagged withhorseradish peroxidase. This conjugate will bind to any IgG antibodiesin the serum that attached to the antigen attached to the microtiterwell. Following another series of washes to remove any unboundconjugate, enzyme substrate is added to each microtiter well. A typicalsubstrate would be tetra-methylbenzidine (TMB) and hydrogen peroxide; inthe presence of horseradish peroxidase, this substrate produces a bluishpurple color. Thus, color only develops when a “sandwich” consisting ofplastic-bound antigen, serum-derived antibody, and enzyme-taggedconjugate has been built up; if the serum contains no antigen-specificantibody molecules, then the enzyme-tagged conjugate will not bind, andno color will develop because the enzyme is absent from the system.After a defined amount of time, a solution of dilute hydrochloric acid,or some other acid, is added to lower the pH of the solution in thewells. At a lower pH, the bluish purple color changes to a yellow color.The absorbance (also called optical density) of the yellow color is thenquantitated using a spectrophotometric instrument. The microtiter wellplate is placed inside the instrument, and the optical density for eachwell (i.e., each sample) is measured at a light wavelength of 450nanometers. The optical density value is directly proportional to theintensity of the yellow color, which in turn is directly proportional tothe amount of antigen-specific IgG antibody bound to the well.

EXAMPLE 1

[0077] Detection of IgG Antibodies Recognizing Histoplasma Capsulatum

[0078]Histoplasma capsulatum antigen (culture filtrate containing both Hand M antigens) intended for use in ID assays was purchased from acommercial source, and adjusted to 20 ug/mL in sodium acetate buffercontaining 0.05M periodate (hereafter referred to as oxidized antigen).For comparative purposes, a duplicate antigen suspension in sodiumacetate buffer without periodate was prepared (hereafter referred to asnonoxidized antigen). After 2 hours at room temperature, the antigenpreparations were diluted 20-fold in PBS to a final concentration of 1ug/mL, then added to microtiter wells (0.1 mL per well). Thus, somemicrotiter wells received oxidized Histoplasma antigen, whereas otherwells received nonoxidized Histoplasma antigen. After an overnightincubation in the refrigerator, the Histoplasma antigen preparations(oxidized or nonoxidized) were discarded from the wells, and PBScontaining 0.1% (w/v) BSA and 5% (w/v) trehalose was added to the wellsas blocking agent. After 2 hours at room temperature, the blocking agentwas discarded, and the plates were then air-dried at room temperaturefor 2 hours. After pouching, the plates were stored in the refrigerator.

[0079] On the day of assay, Histoplasma-coated microtiter wells (somecoated with oxidized antigen, others coated with nonoxidized antigen)were allowed to come to room temperature. During this step, 80 serumsamples previously tested for Histoplasma antibodies by ID (16 positive,64 negative) were diluted 1:400 in sample dilution buffer (PBSTcontaining BSA). Once the wells had reached room temperature(approximately 1 hour), they were washed once with PBST, and the washsolution discarded. Diluted serum samples were then added to duplicatemicrotiter wells (one containing oxidized Histoplasma antigen, the othercontaining nonoxidized Histoplasma antigen). The microtiter wells werecovered with adhesive tape and incubated at room temperature for 1.5hours. Following 4 washes with PBST, each well then received 0.1 mL ofhorseradish peroxidase-conjugated goat anti-human IgG (diluted 1:10000in PBST), and incubation was continued for 1 hour at room temperature.Following another series of 4 washes, each well then received 0.1 mL oftetra-methylbenzidine; after 10 minutes at room temperature, thereaction was stopped by the addition of 0.1N hydrochloric acid. Theoptical density was measured at 450 nM using a spectrophotometric ELISAreader.

[0080]FIG. 1 presents the optical density (OD) values obtained followingincubation of ID-positive and ID-negative serum samples in microtiterwells coated with nonoxidized and oxidized Histoplasma antigen. Whenusing nonoxidized Histoplasma antigen as substrate, the distribution ofOD values for the ID-negative serum group was similar to that observedfor the ID-positive serum group. In contrast, when using the oxidizedHistoplasma antigen as substrate, the distribution of values for theID-negative serum group was markedly different from the distribution ofvalues for the ID-positive serum group. When using the oxidized antigen,16 of 16 (100%) ID-positive serum samples exhibited OD values>1.45,whereas only 4 of 64 (6%) ID-negative serum samples exhibited ODvalues>1.45. Expressed another way, the Histoplasma IgG assay usingoxidized Histoplasma antigen as substrate was 100% sensitive and 94%specific (see Table 2) using an OD cutoff value of 1.45. TABLE 2Specificities of fungal IgG ELISAs using oxidized versus nonoxidizedantigens under conditions giving the same sensitivities ELISA Antigentype Cutoff OD Sensitivity (%) Specificity (%) Histoplasma Oxidized 1.45100 94 IgG Nonoxidized 0.50 100 25 Coccidioides Oxidized 1.00 90 97 IgGNonoxidized 0.22 90 28 Aspergillus Oxidized 0.85 89 95 IgG Nonoxidized0.23 89 65 Blastomyces Oxidized 0.40 100 100 IgG Nonoxidized 0.50 100 87

[0081] For comparative purposes, we determined the specificity of theHistoplasma IgG assay using nonoxidized antigen under conditions wherethe sensitivity using nonoxidized antigen matched that seen usingoxidized antigen (i.e., 100%). Thus, the cutoff OD giving 100%sensitivity when using nonoxidized antigen was 0.50 (FIG. 1). Based onthis cutoff OD value, the specificity of the Histoplasma IgG assay usingnonoxidized antigen was only 25% (see Table 2). Thus, under conditionsproviding 100% sensitivity, oxidation of Histoplasma antigen profoundlyimproved the specificity of an ELISA for detecting Histoplasma IgG.

EXAMPLE 2

[0082] Detection of IgG Antibodies Recognizing Coccidioides immitis

[0083]Coccidioides immitis antigen (culture filtrate containing Fantigen) intended for use in ID assays was purchased from a commercialsource. Microtiter well preparation and assay performance was the sameas described in Example 1 for Histoplasma, with the exception thatconjugate was used at a final dilution of 1:5000 rather than 1:10000.The serum panel evaluated for Coccidioides IgG consisted of 80 samplespreviously tested for Coccidioides antibodies by ID (41 positive, 39negative).

[0084]FIG. 2 presents the optical density (OD) values obtained followingincubation of ID-positive and ID-negative serum samples in microtiterwells coated with nonoxidized and oxidized Coccidioides antigen. Whenusing nonoxidized Coccidioides antigen as substrate, the distribution ofOD values for the ID-negative serum group was similar to that observedfor the ID-positive serum group. In contrast, when using the oxidizedCoccidioides antigen as substrate, the distribution of values for theID-negative serum group was markedly different from the distribution ofvalues for the ID-positive serum group. When using the oxidized antigen,37 of 41 (90%) ID-positive serum samples exhibited OD values>1.00,whereas only 1 of 39 (3%) ID-negative serum samples exhibited ODvalues>1.00. Expressed another way, the Coccidioides IgG assay usingoxidized Coccidioides antigen as substrate was 90% sensitive and 97%specific (see Table 2) using an OD cutoff value of 1.00.

[0085] For comparative purposes, we determined the specificity of theCoccidioies IgG assay using nonoxidized antigen under conditions wherethe sensitivity using nonoxidized antigen matched that seen usingoxidized antigen (i.e., 90%). Thus, the cutoff OD giving 90% sensitivitywhen using nonoxidized antigen was 0.22 (FIG. 2). Based on this cutoffOD value, the specificity of the Coccidioides IgG assay usingnonoxidized antigen was only 28% (see Table 2). Thus, under conditionsproviding 90% sensitivity, oxidation of Coccidioides antigen profoundlyimproved the specificity of an ELISA for detecting Coccidioides IgG.

EXAMPLE 3

[0086] Detection of IgG Antibodies Recognizing Aspergillus fumigatus

[0087]Aspergillus fumigatus antigen (culture filtrate) intended for usein ID assays was purchased from a commercial source. Microtiter wellpreparation and assay performance was the same as described in Example 1for Histoplasma, with the exception that conjugate was used at a finaldilution of 1:5000 rather than 1:10000. The serum panel evaluated forAspergillus IgG consisted of 29 samples previously tested forAspergillus antibodies by ID (9 positive, 20 negative).

[0088]FIG. 3 presents the optical density (OD) values obtained followingincubation of ID-positive and ID-negative serum samples in microtiterwells coated with nonoxidized and oxidized Aspergillus antigen. For bothnonoxidized and oxidized antigen, the overall distribution of OD valuesfor the ID-negative group was visually distinct from the distribution ofOD values for the ID-positive group. However, the range of differencebetween the ID-negative distribution and the ID-positive distributionwas more marked when using oxidized Aspergillus antigen. When using theoxidized antigen, 8 of 9 (89%) ID-positive serum samples exhibited ODvalues>0.85, whereas only 1 of 20 (5%) ID-negative serum samplesexhibited OD values>0.85. Expressed another way, the Aspergillus IgGassay using oxidized Aspergillus antigen as substrate was 89% sensitiveand 95% specific (see Table 2) using an OD cutoff value of 0.85.

[0089] For comparative purposes, we determined the specificity of theAspergillus IgG assay using nonoxidized antigen under conditions wherethe sensitivity using nonoxidized antigen matched that seen usingoxidized antigen (i.e., 89%). Thus, the cutoff OD giving 89% sensitivitywhen using nonoxidized antigen was 0.23 (FIG. 3). Based on this cutoffOD value, the specificity of the Aspergillus IgG assay using nonoxidizedantigen was 65% (see Table 2). Thus, under conditions providing 89%sensitivity, oxidation of Aspergillus antigen markedly improved thespecificity of an ELISA for detecting Aspergillus IgG.

EXAMPLE 4

[0090] Detection of IgG Antibodies Recognizing Blastomyces dermatitidis

[0091]Blastomyces dermatitidis antigen (culture filtrate containing Aantigen) intended for use in ID assays was purchased from a commercialsource. Microtiter well preparation and assay performance was exactlythe same as described in Example 1 for Histoplasma. The serum panelevaluated for Blastomyces IgG consisted of 16 samples previously testedfor Blastomyces antibodies by ID (1 positive, 15 negative).

[0092]FIG. 4 presents the optical density (OD) values obtained followingincubation of ID-positive and ID-negative serum samples in microtiterwells coated with nonoxidized and oxidized Blastomyces antigen. Whenusing the oxidized antigen, the single ID-positive serum sampleexhibited an OD value>0.4, whereas only 0 of 15 (0%) ID-negative serumsamples exhibited OD values>0.40. Expressed another way, the BlastomycesIgG assay using oxidized Blastomyces antigen as substrate was 100%sensitive and 100% specific (see Table 2) using an OD cutoff value of0.40.

[0093] For comparative purposes, we determined the specificity of theBlastomyces IgG assay using nonoxidized antigen under conditions wherethe sensitivity using nonoxidized antigen matched that seen usingoxidized antigen (i.e., 100%). Thus, the cutoff OD giving 100%sensitivity when using nonoxidized antigen was 0.50 (FIG. 4). Based onthis cutoff OD value, the specificity of the Blastomyces IgG assay usingnonoxidized antigen was 87% (see Table 2). Thus, under conditionsproviding 100% sensitivity, oxidation of Blastomyces antigen improvedthe specificity of an ELISA for detecting Blastomyces IgG.

[0094] The above examples are included for illustrative purposes onlyand are not intended to limit the scope of the invention. Manyvariations to those described above are possible. Since modificationsand variations to the examples described above will be apparent to thoseof skill in this art, it is intended that this invention be limited onlyby the scope of the appended claims.

What is claimed is:
 1. A method for producing a fungal antigen suitablefor testing for an antibody to a fungus, the method comprising: a)providing a fungal antigen as a culture filtrate, the fungal antigenhaving not been purified by ion exchange chromatography or isoelectricfocusing from the culture filtrate; and b) contacting the fungal antigenwith an oxidizing agent to produce an oxidized fungal antigen suitablefor testing an antibody to the fungus.
 2. The method of claim 1, whereinthe fungus is selected from the group consisting of a mitosporicTrichocomaceae, an Onygenaceae and a mitosporic Onygenale.
 3. The methodof claim 1, wherein the fungus is selected from the group consisting ofAspergillus, Blastomyces (Ajellomyces), Coccidioides, Histoplasma,Blastocystis and Candida.
 4. The method of claim 3, wherein theAspergillus is Aspergillus fumigatus.
 5. The method of claim 3, whereinthe Blastomyces is Blastomyces dermatitidis.
 6. The method of claim 3,wherein the Coccidioides is Coccidioides immitis.
 7. The method of claim3, wherein the Histoplasma is Histoplasma capsulatum or Histoplasmaduboifi.
 8. The method of claim 1, wherein the fungal antigen has notbeen purified from the culture filtrate.
 9. The method of claim 1,further comprising a step of concentrating the culture filtrate.
 10. Themethod of claim 1, further comprising a step of enzymaticallydeglycosylating the fungal antigen.
 11. The method of claim 1, whereinthe oxidizing agent inactivates the cross-reactive carbohydrate moietyof the fungal antigen but does not disturb the structural integrity orantigenicity of the non-carbohydrate moieties.
 12. The method of claim11, wherein the fungal antigen is a glycosylated protein or peptide andthe oxidizing agent inactivates the cross-reactive carbohydrate moietyof the fungal antigen but does not disturb the structural integrity orantigenicity of the proteineous or peptidyl moiety.
 13. The method ofclaim 1, wherein the oxidizing agent is periodate.
 14. The method ofclaim 1, further comprising a step of attaching the oxidized fungalantigen to a surface of a device suitable for testing an antibody to afungus.
 15. The method of claim 14, wherein the device is selected fromthe group consisting of a microtiter plate, a glass slide, anitrocellulose membrane, a latex bead, a cell, a test tube, a plasticbead, a colloidal gold particle, a colored particle, a magnetic bead anda quantum dot.
 16. A fungal antigen suitable for testing an antibody toa fungus, which antigen is produced by the method of claim
 1. 17. Adevice suitable for testing an antibody to a fungus, which device isproduced by the method of claim
 14. 18. A method of testing an antibodyto a fungus in a sample, the method comprising: a) producing a fungalantigen suitable for testing an antibody to a fungus, comprisingproviding a fungal antigen as a culture filtrate, the fungal antigenhaving not been purified by ion exchange chromatography or isoelectricfocusing from the culture filtrate, and contacting the fungal antigenwith an oxidizing agent to produce an oxidized fungal antigen suitablefor testing an antibody to the fungus; b) contacting a sample suspectedof containing an antibody to a fungus with the oxidized fungal antigenproduced in step a) under suitable conditions to allow binding of theantibody, if present in the sample, to the oxidized fungal antigen; andC) assessing binding between the antibody and the oxidized fungalantigen to determine the presence and/or amount of the antibody in thesample.
 19. The method of claim 18, wherein the fungus is a pathogenicfungus.
 20. The method of claim 18, wherein the sample is a clinicalsample.
 21. The method of claim 20, wherein the clinical sample is ahuman clinical sample.
 22. The method of claim 18, wherein the fungalantigen has not been purified from the culture filtrate.
 23. The methodof claim 18, wherein the oxidizing agent is periodate.
 24. The method ofclaim 18, further comprising a step of attaching the oxidized fungalantigen to a surface of a device suitable for testing an antibody to afungus before contacting the antigen with the sample.
 25. The method ofclaim 18, wherein the binding between the antibody and the oxidizedfungal antigen is assessed by a sandwich or competitive assay format.26. The method of claim 18, wherein the binding between the antibody andthe oxidized fungal antigen is assessed by a format selected from thegroup consisting of an enzyme-linked immunosorbent assay (ELISA),immunoblotting, immunoprecipitation, radioimmunoassay (RIA),immunostaining, latex agglutination, indirect hemagglutination assay(IHA), complement fixation, indirect immunofluorescent assay (IFA),nephelometry, flow cytometry assay, chemiluminescence assay, lateralflow immunoassay, u-capture assay, inhibition assay and avidity assay.27. The method of claim 18, wherein the binding between the antibody andthe oxidized fungal antigen is assessed by an ELISA format.
 28. A methodfor producing an antibody to an fungal antigen, the method comprising:a) producing a fungal antigen comprising providing a fungal antigen as aculture filtrate, the fungal antigen having not been purified by ionexchange chromatography or isoelectric focusing from the culturefiltrate, and contacting the fungal antigen with an oxidizing agent toproduce an oxidized fungal antigen; b) delivering, to a vertebrate ortissue culture, the oxidized fungal antigen, in an amount sufficient toinduce detectable production of an antibody to the antigen; and c)recovering the antibody from the vertebrate or tissue culture.
 29. Themethod of claim 28, wherein the oxidizing agent is periodate.
 30. Themethod of claim 28, wherein the vertebrate is a non-human mammal.
 31. Anantibody to a fungal antigen, which antibody is produced by the methodof claim
 28. 32. The antibody of claim 31, which is a polyclonalantiserum.
 33. A method for producing a monoclonal antibody to a fungalantigen, the method comprising: a) producing a fungal antigen comprisingproviding a fungal antigen as a culture filtrate, the fungal antigenhaving not been purified by ion exchange chromatography or isoelectricfocusing from the culture filtrate, and contacting the fungal antigenwith an oxidizing agent to produce an oxidized fungal antigen; b)delivering, to a vertebrate or tissue culture, the oxidized fungalantigen, in an amount sufficient to induce detectable production of anantibody to the antigen; c) removing at least a portion ofantibody-producing cells from the vertebrate or tissue culture; d)immortalizing the removed antibody-producing cells; e) propagating theimmortalized antibody-producing cells; and f) harvesting monoclonalantibody produced by the immortalized antibody-producing cells.
 34. Anmonoclonal antibody to a fungal antigen, which monoclonal antibody isproduced by the method of claim
 33. 35. A hybridoma capable of producinga monoclonal antibody to a fungal antigen, the hybridoma is produced bysteps a)-d) of the method of claim
 33. 36. A method of testing for afungal antigen in a sample, the method comprising: a) providing ananti-fungal polyclonal antiserum produced by the method of claim 32; b)contacting a sample suspected of containing a fungal antigen with thepolyclonal antiserum under suitable conditions to allow binding of thefungal antigen, if present in the sample, to the polyclonal antiserum;and c) assessing binding between the fungal antigen and the polyclonalantiserum to determine the presence and/or amount of the fungal antigenin the sample.
 37. A method of testing for a fungal antigen in a sample,the method comprising: a) providing an anti-fungal antigen monoclonalantibody produced by the method of claim 35; b) contacting a samplesuspected of containing a fungal antigen with the monoclonal antibodyunder suitable conditions to allow binding of the fungal antigen, ifpresent in the sample, to the monoclonal antibody; and c) assessingbinding between the fungal antigen and the monoclonal antibody todetermine the presence and/or amount of the fungal antigen in thesample.
 38. A device for simultaneously testing for a plurality offungal antibodies, the device comprising a plurality of fungal antigensproduced by the method of claim 1 attached to a surface of the devicesuitable for testing an antibody to a fungus, wherein the plurality offungal antigens are attached to areas of the surface that are physicallydistinct from each other.
 39. The device of claim 38, which devicecomprises an immunoblot and wherein the plurality of fungal antigens areattached to separate stripes in the immunoblot.
 40. The device of claim38, which device comprises an IFA well and wherein the plurality offungal antigens are attached to different spots within the IFA well. 41.The device of claim 38, which device comprises an IFA slide and whereinthe plurality of fungal antigens are attached to different wells of theIFA slide.
 42. The device of claim 38, which is selected from the groupconsisting of a microtiter plate, a glass slide, a nitrocellulosemembrane, a latex bead, a cell, a test tube, a plastic bead, a colloidalgold particle, a colored particle, a magnetic bead and a quantum dot.43. A device for testing for a fungal antigen, the device comprising ananti-fungal antibody produced by the method of claim 28 attached to asurface of the device.
 44. A device for testing for a fungal antigen,the device comprising an anti-fungal monoclonal antibody produced by themethod of claim 33 attached to a surface of the device.
 45. A device forsimultaneously testing for a plurality of fungal antigens, the devicecomprising a plurality of antibodies produced by the method of claim 28attached to a surface of the device suitable for testing for a fungalantigen, wherein the plurality of fungal antibodies are attached toareas of the surface that are physically distinct from each other.
 46. Adevice for simultaneously testing for a plurality of fungal antigens,the device comprising a plurality of monoclonal antibodies produced bythe method of claim 33 attached to a surface of the device suitable fortesting for a fungal antigen, wherein the plurality of fungal antibodiesare attached to areas of the surface that are physically distinct fromeach other.